CN103360484B - Upland cotton protein GhMADS22, and coding gene and application thereof - Google Patents

Abstract

The invention discloses an upland cotton GhMADS22 protein, and a coding gene and application thereof. The protein GhMADS22 has one of the following amino acid residue sequences: 1) amino acid residue sequence disclosed as SEQ ID No.2 in the sequence table; and 2) 1) derived protein related to plant photosynthetic efficiency, which is subjected to substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid residue sequence disclosed as SEQ ID No.2 in the sequence table. The protein and coding gene thereof can be used for cultivating cotton with advanced reproductive growth, higher planting density or increased number of secondary bolls, and enhancing the cotton yield, thereby laying foundation for cultivation of transgenic plants.

Description

A kind of upland cotton Protein G hMADS22 and encoding gene thereof and application

Technical field

The invention belongs to biological technical field, be specifically related to a kind of upland cotton albumen and encoding gene thereof and application.

Background technology

Cotton is one of most important cash crop of China, in national product, occupy critical role, but due to grain and cotton strive ground, to a certain degree limiting the development of cotton.The selection and popularization of short season cotton kind, can alleviate the contradiction that ground striven by grain and cotton, is the effective way realizing reaping a bumper harvest of grain and cotton.But there is the inadequate problem of prematureness in short season cotton at present.

Prematureness, as one of the important character of cotton, becomes the breeding objective that upland cotton is important.Research shows, blooms sooner or later closely related with the prematureness of cotton.Therefore clone genes involved of blooming, expression analysis and transgenosis functional verification are carried out to it, for short season cotton breeding provides the genetic resources of high-quality.

Summary of the invention

The invention provides a kind of Protein G hMADS22 and encoding gene and its thereof to apply, described Protein G hMADS22 derives from cotton (Gossypium spp).

Albumen of the present invention is following 1) or 2) albumen:

1) protein of the composition of the aminoacid sequence shown in SEQ ID № .2 in sequence table;

2) by the amino acid residue sequence of the SEQ ID № .2 in sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to flowering of plant by 1) protein that derives.

In sequence table, the aminoacid sequence shown in SEQ ID № .2 is made up of 249 amino-acid residues.

Above-mentioned 1) and 2) in GhMADS22 albumen can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned 1) and 2) in the encoding gene of GhMADS22 albumen by the DNA sequence dna shown in the 1-747 position Nucleotide of SEQ ID № .1 in sequence table being lacked the codon of one or several amino-acid residue, and/or to obtain after the missense mutation carrying out one or several base pair.

The nucleic acid molecule of described GhMADS22 albumen of encoding also belongs to protection scope of the present invention.

Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA, hnRNA or tRNA etc.

When described nucleic acid molecule is DNA, it has one of following nucleotide sequence:

1) nucleotide sequence of SEQ ID №: 1 1-747 position in sequence table;

2) polynucleotide sequence of SEQ ID №: 2 protein sequence in polynucleotide;

3) nucleotide sequence that the DNA sequence dna that can limit with SEQ ID in sequence table №: 1 under high high stringency conditions is hybridized;

4) with 1) or 2) or 3) DNA sequence dna that limits has more than 70% homology, and coding identical function protein DNA sequence; Concrete, described homology is more than 75%; Concrete is more than 80% again; Concrete is more than 85% again; Concrete is more than 90% again; Concrete is more than 95% again; Concrete is more than 96% again; Concrete is more than 97% again; Concrete is more than 98% again; Concrete is more than 99% again.

Above-mentioned high high stringency conditions can be the solution with 6 × SSC, 0.5%SDS, and hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Wherein, SEQ ID №: 1 in sequence table is made up of 879 Nucleotide, its open reading frame (ORF) is from 5 ' end 1-747 position Nucleotide, the protein (GhMADS22 albumen) in polynucleotide shown in SEQ ID №: 2.

Recombinant vectors containing above-mentioned nucleic acid molecule, expression cassette, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.

Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.

Described recombinant expression vector can use existing expression vector establishment.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any one enhancement type, composing type, organizing specific type or inducible promoter before its transcription initiation Nucleotide, they can be used alone or are combined with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer.For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, as being added in enzyme or the gene (gus gene, GFP gene, luciferase genes etc.) of luminophor, the antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance or the chemical resistance reagent marker gene (as anti-weedkiller gene) etc. expressing in plant and can produce colour-change.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.

The primer pair of encoding gene total length of the present invention or its any fragment of increasing also belongs to the scope of protection of the invention.

The nucleotides sequence of a primer in described primer pair is classified as the nucleotide sequence of SEQ ID №: 3 in sequence table, and the nucleotides sequence of another primer in described primer pair is classified as the nucleotide sequence of SEQ ID №: 4 in sequence table.

Another object of the present invention be to provide albumen of the present invention, encoding gene and containing the recombinant vectors of described encoding gene, expression cassette, transgenic cell line or recombinant bacterium following 1)-8) application at least one

1) reproductive growth of plant is made in advance;

2) output is improved;

3) regulating plant bolting;

4) regulating plant lotus throne number of sheets amount;

5) regulating plant stem leaf quantity

6) regulating plant inflorescence types;

7) variation of regulating plant floral organ;

8) the coming off of regulating plant floral organ.

Described regulating plant bolting is for promoting plant bolting;

Described regulating plant lotus throne number of sheets amount is for reducing plant lotus throne number of sheets amount;

Described regulating plant stem leaf quantity is that minimizing axis leave quantity and stem leaf change the bract being similar to stem leaf into;

Described regulating plant inflorescence types is that inflorescence changes solitary flower or terminal inflorescence into;

Described regulating plant floral aberrance is for form secondary flower at bract armpit place or gynoecium base portion;

Described regulating plant floral organ come off for postpone floral organ come off;

Described plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana or cotton.

Further object of the present invention is to provide a kind of method of cultivating transgenic plant.

Encoding gene of the present invention is specifically imported object plant by the method for cultivation transgenic plant of the present invention, obtains transgenic plant; Described transgenic plant, compared with described object plant, have following 1)-8) middle at least one phenotype:

1) reproductive growth of described transgenic plant is early than described object plant;

2) output of described transgenic plant is higher than described object plant;

3) the bolting time of described transgenic plant is early than described object plant;

4) the lotus throne number of sheets amount of described transgenic plant is less than described object plant;

5) the stem leaf quantity of described transgenic plant is less than described object plant and changes the bract being similar to stem leaf into;

6) described transgenic plant are compared to described object plant, and inflorescence changes solitary flower or terminal inflorescence into;

7) floral organ of described transgenic plant is compared to described object plant, forms secondary flower at bract armpit place or gynoecium base portion;

8) described transgenic plant floral organ comes off and is later than described object plant.

In described method, described by encoding gene importing object plant of the present invention, import in object plant particular by the recombinant vectors containing described encoding gene of the present invention.

In described method, described plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana or cotton.

Present invention finds a kind of new gene GhMADS22, carried out transgenosis, obtain the bolting time advance of transgenic plant, simultaneously plant lotus throne number of sheets amount and reduce, stem leaf quantity also reduces; And inflorescence changes solitary flower, top flower into; Secondary flower is formed, and the floral organ time that comes off also postpones, and therefore utilizes this gene to can be used to cultivate the cotton that reproductive growth shifts to an earlier date, planting density is higher or secondary bell number is more, to increase output of cotton, for the cultivation of transgenic plant is laid a good foundation.

Accompanying drawing explanation

Fig. 1 is the expression of GhMADS22 gene at terminal bud different development stage, wherein, in Fig. 1 0SAM, 1SAM, 2SAM, 3SAM represent respectively cotyledon flatten time, first, second and third sheet true leaf flatten time terminal bud.

Fig. 2 is the expression of GhMADS22 gene at different tissues.

Fig. 3 is the structural representation of the plant expression vector pBI121-GhMADS22 inserting GhMADS22 gene coded sequence.

Fig. 4 is transfer-gen plant Molecular Identification figure, wherein Fig. 4 A be numbered 1,2,3,44 strain T ₀in generation, turns the qualification figure of GhMADS22 Arabidopis thaliana on DNA level, and WT is wildtype Arabidopsis thaliana contrast; Fig. 4 B is isozygoty the qualification figure of Arabidopsis plant on rna level, the WT that the positive that 4 strains are numbered 3,22,31,44 turns GhMADS22 is wildtype Arabidopsis thaliana contrast.

Fig. 5 is the phenotype comparison diagram of the transgenic arabidopsis of wild-type and process LAN GhMADS22, and wherein, A: WT lines, has indeterminate growth inflorescence; B: transfer-gen plant, form top flower and solitary flower, stem leaf changes the bract of similar leaf into; C: wild-type inflorescences, indeterminate growth inflorescence; D: wild-type flower, 4 sepals, 4 petals, 6 stamens, 1 gynoecium; E-J: the flower of transfer-gen plant, wherein, (E): 3 petals, the stamen of 8 random arrangements; (F): 7 petals, 7 stamens; 2 gynoeciums; (G, H): without petal; (G-J): one or two secondary flower; (J): floral organ postpones to come off.

embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Examination material is supplied to be Shine Early cotton kind CCRI 36(CCRI36) (purchased from the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute), planted in 2012 in experimental plot, old portion of institute of the Chinese Academy of Agricultural Sciences, in in May, 2012 get cotyledon flatten time, first true leaf flatten time, second true leaf flatten time, the 3rd true leaf flatten time terminal bud, the different tissues such as root, stem, leaf, bract, sepal, petal, stamen, carpel, ovule, fiber, terminal bud are got in July, 2012, put into the liquid nitrogen container filling liquid nitrogen immediately, deposit for subsequent use in-80 DEG C of refrigerators after taking back laboratory.

Cloning vector is -T Easy Vector, purchased from Promega company.

Bacterial classification is that bacillus coli DH 5 alpha is purchased from Tian Gen Bioisystech Co., Ltd.

Taq archaeal dna polymerase is purchased from TaKaRa company, DNA glue reclaims test kit purchased from Shanghai Sheng Gong Bioisystech Co., Ltd, the microbiotic such as Amp, X-gal, IPTG, Rif, Streptomycin sulphate, Kan are Sigma Products, RNA extracts test kit purchased from Tian Gen Bioisystech Co., Ltd, SuperScriptTM III First-Strand Synthesis System for RT-PCR kit is purchased from invitrogen company, and all the other reagent are import or domestic analytical reagent.

The preparation of embodiment 1, cotton gene GhMADS22

1, the extraction of RNA

Carry out according to test kit operation instructions, concrete steps are as follows:

Before operation, first preparation contains the lysate SL of final concentration 5% mercaptoethanol, as added 50 μ l beta-mercaptoethanols in 950 μ l SL.

1). homogenized:

100mg CCRI 36 (CCRI36) plant leaf is rapid grind into powder in liquid nitrogen, add the lysate SL (please first check whether before use and added beta-mercaptoethanol) that 500 μ l prepare, vortex concuss mixes, and room temperature places 5min.

2). all solution is transferred to (Filter column CS is placed in collection tube) on Filter column CS, 12,000rpm (~ 13,400 × g) centrifugal 2 minutes, supernatant in careful absorption collection tube is in centrifuge tube without RNA enzyme (RNase-free) of new 1.5ml, and suction nozzle is avoided contacting the pellet cell debris in collection tube as far as possible.

3). slowly add the dehydrated alcohol of 0.4 times of supernatant volume, mixing (now may occur precipitation), the solution obtained is proceeded in adsorption column CR3 together with precipitation, 12,000rpm (~ 13,400 × g) centrifugal 30 seconds, outwell the waste liquid in collection tube, adsorption column CR3 is put back in collection tube.

4). in adsorption column CR3, add 350 μ l protein liquid removal RW1,12,000rpm (~ 13,400 × g) centrifugal 30-60 second, outwell the waste liquid in collection tube, adsorption column CR3 is put back in collection tube.

5) preparation of .DNase I working fluid: get 10 μ l DNase I storage liquid and put into new RNase-free centrifuge tube, add 70 μ l RDD solution, softly mix.

6). add the DNase I working fluid of 80 μ l to adsorption column CR3 central authorities, room temperature places 15 minutes.

7). in adsorption column CR3, add 350 μ l protein liquid removal RW1,12,000rpm (~ 13,400 × g) centrifugal 30-60 second, outwell the waste liquid in collection tube, adsorption column CR3 is put back in collection tube.

8). in adsorption column CR3, add 700 μ l rinsing liquid RW (please first check whether before use and added ethanol), room temperature leaves standstill 2 minutes, and 12,000rpm (~ 13,400 × g) centrifugal 30-60 second, outwell the waste liquid in collection tube, adsorption column CR3 is put back in collection tube.

9). in adsorption column CR3, add 500 μ l rinsing liquid RW (please first check whether before use and added ethanol), room temperature leaves standstill 2 minutes, and 12,000rpm (~ 13,400 × g) centrifugal 30-60 second, outwell the waste liquid in collection tube, adsorption column CR3 is put back in collection tube.

10) centrifugal 2 minutes of .12,000rpm (~ 13,400 × g), outwells waste liquid.Adsorption column CR3 is placed in room temperature and places several minutes, thoroughly to dry rinsing liquid remaining in sorbing material.

11). adsorption column CR3 is put into a new RNase-free centrifuge tube, the unsettled dropping in the middle part to adsorption film 30-100 μ l RNase-free ddH2O, room temperature places 2 minutes, 12, centrifugal 2 minutes of 000rpm (~ 13,400 × g), obtains RNA solution.

2, the preparation of cDNA

Use the SuperScript of Invitrogen company ^tMiII First-Strand Synthesis System forRT-PCR, will often kind of of short duration centrifugal mixing of mixture before using.

1) centrifuge tube of the 0.5ml of DEPC process is put on ice, adds in the following order:

The total serum IgE 5ug that step 1 prepares

50uM Oligo(dT) ₂₀1ul

10mM dNTP Mix 1ul

Supply 10ul with DEPC process water, be made into RNA/Primer mixture;

2) 65 DEG C of insulation 5min, ice bath is 1min at least;

3) in the centrifuge tube of the 0.5ml of DEPC process, prepare cDNA Synthesis Mix:

4) 10ul cDNA Synthesis Mix is added in RNA/Primer mixture, softly mixes, of short duration centrifugal;

5) 50 DEG C of insulation 50min; Put on ice after 85 DEG C of insulation 5min, the centrifugal several seconds collects reaction solution;

6) 1ulRNaseH is added in reaction solution, 37 DEG C of insulation 20min;

7) get 1ul reverse transcription product electrophoresis detection, all the other-20 DEG C save backup.

3, the amplification of gene

Primer sequence is:

Forward primer: 5 '-ATGGGGAGGGGTAGGGTTCAGTT-3 '

Reverse primer: 5 '-TGGGATTACACATAGATGCAGGTCA-3 '

With the primer of above-mentioned design and the preparation-obtained CCRI 36cDNA of step 2 for template, carry out pcr amplification.

Pcr amplification product is checked order.Sequencing result shows, above-mentioned pcr amplification obtains having the nucleotide sequence of SEQ ID №: 1 in sequence table, 879bp altogether, wherein encode head of district 747bp, in this coding region ordered list, the nucleotide sequence of 1-747 position in SEQ ID №: 1, has the fragment called after GhMADS22 of the nucleotide sequence of 1-747 position in SEQ ID №: 1 in sequence table by this.Amino acid residue sequence shown in SEQ ID №: 2 in this GhMADS22 full-length cDNA polynucleotide.

The functional verification of embodiment 2, cotton gene GhMADS22

One, the spatial and temporal expression pattern analysis of cotton gene GhMADS22

In order to study the expression pattern of GhMADS22 gene, qRT-PCR is used to detect the expression of GhMADS22 in the expression and different tissues of CCRI 36 terminal bud different development stage.

Using the cDNA of the terminal bud and different tissues that dilute CCRI 36 different development stage after 10 times as template, take ACTIN as internal reference, carry out the fluorescence relative quantitative assay of gene GhMADS22.

Amplification gene GhMADS22 primer sequence used is:

Forward primer q22F:5 '-AGGAAATGACCCATCAGCCAC-3 '

Reverse primer q22R:5 '-GCTGCACTAGGATTACCCTCTTC-3 '

ACTIN internal reference primer sequence is:

Forward primer: 5 '-ATCCTCCGTCTTGACCTTG-3 '

Reverse primer: 5 '-TGTCCGTCAGGCAACTCAT-3 '

Concrete operation step is as follows:

1) design fluorescent quantitation primer with Oligo6.0, PCR detects primer specificity;

2) according to following order preparation quantitative fluorescent PCR system:

3 repetitions are set, because application of sample can exist a little error, therefore will according to the number polygamy system of sample some, in case sample is not enough.

3) PCR program:

First 95 DEG C of denaturation 10min, then 95 DEG C of sex change 10s, 60 DEG C of annealing 35s, 72 DEG C extend 1min, 40 circulations, and 72 DEG C extend 10min, and at 72 DEG C, fluorescent signal is collected at 1min place.

4) data analysis

Adopt relative quantification Δ Δ Ct method to analyze, as depicted in figs. 1 and 2, the expression analysis according to terminal bud different development stage finds result, and GhMADS22 raises gradually along with the growth expression amount of terminal bud, illustrates that it has the function promoting bud differentiation; Expression analysis according to different tissues finds, the expression amount of GhMADS22 in blade, terminal bud, bract, sepal, stamen, carpel etc. is higher, illustrates that GhMADS22 may be relevant with the growth of blade and floral organ.

Two, cotton gene GhMADS22 is promoting the application in flowering of plant

(1), the structure of expression vector

With CCRI 36cDNA for template, to be added with the primers F of corresponding restriction enzyme site: 5 '-GC tCTAGAaTGGGGAGGGGTAGGGTTCAGTTG-3 ' (underscore is XbaI enzyme cutting site) (SEQ ID №: 3 see in sequence table); R:5 '-TGCAGGTCACACATCAGTTTGTC-3 ' (SEQ ID №: 4 see in sequence table), amplification obtains the PCR primer comprising GhMADS22 gene coding region, and the nucleotide sequence of PCR primer is shown in shown in the 1-863 position Nucleotide of SEQ ID №: 5 in sequence table; This PCR primer is linked on pGEM-T Easy cloning vector, and transformation of E. coli DH5 α, through the exactness in PCR and sequence verification sequence and direction, correct clone is selected to extract plasmid, cut with XbaI and SacI enzyme, obtain shown in SEQ ID №: 5 that goal gene fragment sequence is shown in sequence table.

Plant expression vector pBI121 is cut through XbaI and SacI enzyme, obtains carrier framework;

Above-mentioned carrier framework is connected with above-mentioned purpose gene fragment T4DNA ligase enzyme.

It is as follows that the enzyme of pBI121 cuts system:

Linked system is as follows:

Spent the night by connection product 4 DEG C, then transform E. coli cells, chooses mono-clonal and carries out single bacterium colony PCR checking and sequence verification, expands and shakes correct mono-clonal extraction plasmid.Gained plasmid is that the fragment of SEQ ID №: 5 1-907 position nucleotide sequence in sequence table is inserted into the carrier obtained between XbaI and SacI of pBI121, and by this plasmid called after pBI121-GhMADS22, its structural representation as shown in Figure 3.

(2) acquisition of GhMADS22 Arabidopis thaliana, is turned

1, the acquisition of recombinational agrobacterium

Plasmid pBI121-GhMADS22 is proceeded in Agrobacterium LBA4404 competent cell, obtains recombinant bacterium.The plasmid extracting recombinant bacterium sends to order-checking, by the recombinant bacterium called after LBA4404/pBI121-GhMADS22 containing plasmid pBI121-GhMADS22 correct for order-checking.

2, transform

(1) plantation of Arabidopis thaliana: first (5min washed by the alcohol of 75%, and 0.1% mercuric chloride washes 3min, sterilizing ddH by wildtype Arabidopsis thaliana seed disinfection ₂o washes 6 times), aseptic filter paper dries, then chosen on MS substratum with aseptic toothpick, with sealed membrane sealing, then wrap masking foil and place 4 DEG C of vernalization, remove masking foil after 2-3 days and transfer them to light temperature incubator (22 DEG C, 16h illumination/8h is dark) cultivate, transplant in soil after growing four leaves, be transferred to (22 DEG C, 16h illumination/8h is dark) on Arabidopis thaliana culturing room culturing rack.

(2) conversion of Arabidopis thaliana: inoculation LBA4404/pBI121-GhMADS22 in 5ml LB substratum (be the kantlex of 50ug/ml containing final concentration), 28 DEG C, 180rpm, shaken overnight; Be transferred in 200mlLB substratum with 1: 50 ratio, 28 DEG C, 180rpm is cultured to OD ₆₀₀=1.2; The centrifugal 15min of 4000rpm collects thalline, thalline is resuspended in permeabilization buffer (sucrose solution of 5% adds the Silwet L-77 of 0.02%), with resuspended penetrating fluid for contrast, regulates OD ₆₀₀=0.8; The pod of the Hua Hejie pollinated by the wild-type Arabidopsis plants of blooming cuts off with scissors, thaliana flower is immersed to be equipped with in the small beaker of permeabilization buffer and contaminates 50sec, then the Arabidopsis plant contaminated is placed dark culturing in large plastic tank, continue to cultivate in Arabidopis thaliana culturing room after 24h; After fruit pod maturation, mix sowing, obtain T ₀for the Arabidopis thaliana seed turning GhMADS22.

By T ₀cultivate on the MS substratum of added with antibiotic kantlex after sterilization vernalization for seed, negative transgenosis strain can not normal growth, and screening obtains positive T ₀in generation, turns GhMADS22 Arabidopis thaliana.

3, the Molecular Identification of transgenic progeny

Extract positive T ₀for the genomic dna turning GhMADS22 Arabidopis thaliana, with forward primer 5 '-TTCATTTGGAGAGAACACGGGGGA-3 ' (mating with one section of nucleotide sequence of 35S promoter on carrier) and reverse primer 5 '-ATTGGAGTTGCGATGTTGTGCTGC-3 ' for primer carries out pcr amplification, with wildtype Arabidopsis thaliana (WT) for contrast.

PCR system (25ul) is as follows:

PCR program is as follows:

1% agarose electrophoresis detects, and as shown in Figure 4 A, 1-4 swimming lane can detect the fragment of 742bp to result respectively, is defined as positive T ₀in generation, turns GhMADS22 Arabidopis thaliana; The genomic dna extracting wild-type (WT) Arabidopis thaliana in contrast, with same primer, does not obtain object fragment.

By positive T ₀in generation, turns GhMADS22 Arabidopis thaliana individual plant sowing, sowing, and screening, until obtain homozygous lines.

The Stochastic choice 4 strain positive turns the Arabidopsis plant that isozygotys of GhMADS22, be numbered 3,22,31,44, over-ground part organized mixing RNA is extracted when blooming, and reverse transcription is cDNA, then with this cDNA for template, carried out the fluorescence relative quantitative assay of gene GhMADS22 by qRT-PCR, further the expression of qualification GhMADS22 in Arabidopis thaliana, wherein, the primer sequence that amplification gene GhMADS22 is used is:

Amplification gene GhMADS22 primer sequence used is:

Forward primer q22F:5 '-AGGAAATGACCCATCAGCCAC-3 '

Reverse primer q22R:5 '-GCTGCACTAGGATTACCCTCTTC-3 '

Reference gene is TUB2, and the primer sequence of amplification reference gene is:

Forward primer: 5 '-CAACTGGATTCAAGTGCGGG-3 '

Reverse primer: 5 '-TTCTCCACCAACTTCCTCATAATC-3 '

Wildtype Arabidopsis thaliana is contrast.As shown in Figure 4 B, GhMADS22 has different expression levels in different strains to result, and does not express in wild-type (WT).

Three, the phenotype of GhMADS22 Arabidopis thaliana is turned

3,22,31,44 isozygoty and turn GhMADS22 Arabidopis thaliana, wildtype Arabidopsis thaliana (WT) and turn the sowing of empty carrier Arabidopis thaliana will be numbered, cultivate under long-day conditions (16h illumination/8h dark).

Turn empty carrier Arabidopis thaliana consistent with wildtype Arabidopsis thaliana phenotype.Add up 4 lotus throne numbers of sheets of isozygotying when turning GhMADS22 strain and wildtype Arabidopsis thaliana bolting time and bolting (because stem leaf changes bract into, therefore do not add up stem leaf number when blooming), experiment in triplicate, each strain is that reference carries out significance analysis with wild-type, use One way ANOVA and Bonferroni in software SigmaStat to test, statistic analysis result is in table 1.In table 1, P represents statistical significant level, and wherein * * represents P < 0.01, * * * and represents P < 0.001;

Table 1

As can be seen from Table 1, under long-day conditions (16h illumination/8h is dark), transfer-gen plant reduces 1-3 sheet than lotus throne leaf during wild-type bolting, and the result of composition graphs 4B, the higher general bolting of strain of known GhMADS22 expression amount also more early, the lotus throne number of sheets is fewer, illustrates and more early enter generative growth phase; And compared with wild-type, the bolting time of each GhMADS22 of turning strain, the difference of the lotus throne number of sheets all reach statistical significant level.Table 1 result shows that GhMADS22 can promote plant bolting, makes the reproductive growth of plant in advance.

Select GhMADS22 expression amount the highest, bolting No. 31 strains the earliest and wildtype Arabidopsis thaliana (WT) carry out Phenotypic Observation, phenotype under long day as shown in Figure 5, A, C, D is wildtype Arabidopsis thaliana (WT), B, E-J is transgenosis No. 31 strains, wild-type is indeterminate growth inflorescence (A, C), transfer-gen plant forms top flower or solitary flower (B), wild-type flower has four sepals, four petals, six stamens, a gynoecium (D), the floral organ of transfer-gen plant has variation in various degree, as: the stamen (E) of three elongated petals and 8 random arrangements, two gynoeciums, 7 petals and stamen (F), there is no petal, the sepal (G) of two variations, there is no petal and sepal (H), one or two secondary flower (G-J) is formed at bract armpit place or gynoecium base portion, floral organ comes off more late (J).Fig. 5 experimental result shows, the adjustable plant inflorescence of GhMADS22, makes indeterminate growth inflorescence change solitary flower and top flower into, thus improves the planting density of plant, and then improve output; GhMADS22 can postpone coming off the time of floral organ, thus improves plant biomass; GhMADS22 also can make bract armpit place or gynobase minister go out secondary flower, thus increases the quantity of flower, improves output.

Claims (12)

1. an albumen is the protein of the composition of the aminoacid sequence shown in SEQ ID No.2 in sequence table.

2. the encoding gene of albumen described in claim 1.

3. encoding gene according to claim 2, is characterized in that: described encoding gene is the Nucleotide of SEQ ID No.1 1-747 position in sequence table.

4. the recombinant vectors containing the encoding gene described in Claims 2 or 3; Described recombinant vectors is specially recombinant expression vector or recombinant cloning vector.

5. the expression cassette containing the encoding gene described in Claims 2 or 3.

6. the recombinant bacterium containing the encoding gene described in Claims 2 or 3.

7. the primer pair of encoding gene total length or its any fragment described in Claims 2 or 3 of increasing, it is characterized in that: the nucleotides sequence of a primer in described primer pair is classified as the nucleotide sequence of SEQ ID No.3 in sequence table, the nucleotides sequence of another primer in described primer pair is classified as the nucleotide sequence of SEQ ID No.4 in sequence table.

8. albumen according to claim 1 or the encoding gene described in Claims 2 or 3 or recombinant vectors according to claim 4 or expression cassette according to claim 5 or recombinant bacterium according to claim 6 are following 1)-8) application at least one:

1) reproductive growth of plant is made in advance;

2) output is improved;

3) regulating plant bolting;

4) regulating plant lotus throne number of sheets amount;

5) regulating plant stem leaf quantity

6) regulating plant inflorescence types;

7) variation of regulating plant floral organ;

8) the coming off of regulating plant floral organ.

9. application according to claim 8, is characterized in that:

Described regulating plant bolting is for promoting plant bolting;

Described regulating plant lotus throne number of sheets amount is for reducing plant lotus throne number of sheets amount;

Described regulating plant stem leaf quantity is that minimizing axis leave quantity and stem leaf change the bract being similar to stem leaf into;

Described regulating plant inflorescence types is that inflorescence changes solitary flower or terminal inflorescence into;

Described regulating plant floral aberrance is for form secondary flower at bract armpit place or gynoecium base portion;

Described regulating plant floral organ come off for postpone floral organ come off;

Described plant is dicotyledons or monocotyledons.

10. according to application according to claim 9, it is characterized in that: described dicotyledons is Arabidopis thaliana or cotton.

11. 1 kinds of methods of cultivating transgenic plant, are that the encoding gene described in Claims 2 or 3 is imported object plant, obtain transgenic plant; Described transgenic plant, compared with described object plant, have following 1)-8) middle at least one phenotype:

1) reproductive growth of described transgenic plant is early than described object plant;

2) output of described transgenic plant is higher than described object plant;

3) the bolting time of described transgenic plant is early than described object plant;

4) the lotus throne number of sheets amount of described transgenic plant is less than described object plant;

5) the stem leaf quantity of described transgenic plant is less than described object plant and changes the bract being similar to stem leaf into;

6) described transgenic plant are compared to described object plant, and inflorescence changes solitary flower or terminal inflorescence into;

7) floral organ of described transgenic plant is compared to described object plant, forms secondary flower at bract armpit place or gynoecium base portion;

8) described transgenic plant floral organ comes off and is later than described object plant.

12. methods according to claim 11, is characterized in that: described by the encoding gene importing object plant described in Claims 2 or 3, import in object plant particular by recombinant vectors according to claim 4.